The present invention, in general, refers to the use of carboxypeptidase protein inhibitors and of their natural variants or of forms redesigned by protein engineering as well as peptidomimetic molecules derived from the former as an anticancerous medicament, and more specifically, to control the invasion and the metastatic growth in tumors and to its use for therapeutic purposes.
Protease inhibitors have been extensively related to processes of biomedical interest, such as the processing of hormone and neuropeptide precursors, inflammatory processes, tumoral processes, etc. (Avilxc3xa9s, F. X., editor of xe2x80x9cInnovations in proteases and their inhibitorsxe2x80x9d, Walter de Gruyter, 1993; Trow, W. and Kennedy, A. R. eds. xe2x80x9cProtease inhibitors as cancer chemopreventive agentsxe2x80x9d; Plenum Publishing Corp., 1993; Kennedy, A. R. (1994) Cancer Res., 54: 1995s-2005s). Among the protease inhibitors are the metalocarboxypeptidase inhibitors. One of them, the inhibitor from potato (from now on, referred to as xe2x80x9cPCIxe2x80x9d) has been extensively studied, from the structural and functional point of view, as an inhibitor by our research team (Molina, M. A. et al. (1992) Gene, 116: 129-138; Oliva, B. et al. (1991) Biochem. Biophys. Res. Comm., 176: 616-621 and 627-632; Tapia et al. (1991) J. Mol. Engineer., 1: 249-266; Querol, E. et al. (1993) in xe2x80x9cInnovations in Proteases and their Inhibitorsxe2x80x9d (F. X. Avilxc3xa9s, ed.) : 447-494. Walter de Gruyter, Berlin, 1993; Molina M. A. et al. (1994) J. Biol. Chem., 269: 22087-22094; Marino-Busjle, C. et al. (1994) Applied Microb. and Biotech., 41: 632-637; Oliva, B. et al. (1995) J. Mol. Model., 5:1-15).
A relevant structural feature of the PCI is that it is a small protein which contains a globular core stabilized by means of three disulfide bridges which constitute a cystine knot. We are referring to a topological pattern shared by other proteins, whether functionally related or not, which employ various cysteines concentrated in a central zone in order to create a stabilizing knot of disulfide bridges. This ensemble of proteins constitutes the so called group of knotins, because of the topological knot they form. We have to underline that, among the proteins which share this structural pattern with PCI, there are various cellular growth factors such as, for instance, the a and xcex2-TGF, NGF, PDGF, EGF and those of the insulin family (Isaacs, N. (1995) Curr. Opin. Struct. Biol., 5:391-395; Lin, S et al. (1995) Nature Struct. Biol., 2, 835-837; Sun P. and Davies, D. (1995) Ann. Rev. Biphys. Biolmol. Struct., 24: 269-291). In the specific case of the PCI, the globular core contains 27 amino acid residues and is flanked by two tails of 7 residues (the N-terminal tail) and of 5 residues (the C-terminal tail). The disulfide bridges are between cysteines 8-24, 12-27 and 18-34. This is the most frequent natural form, the so called IIa form, although there are other isoforms (Hass, G. M. and Ryan, C. A. (1981) Meth. Enzymol., 80: 778-791).
A great number of studies have described the expression of proteases and peptidases in different types of tumoral cells (Chen, W. T. (1992) Curr. Opin. Cell. Biol., 4: 802-809; Birkedal-Hansen, H. (1995) Curr. Opin. Cell. Biol., 7: 728-735). Some of these proteases are related to the processing of peptidic hormones, growth factors, etc. For instance, the existence of proteases responsible for the mobilization of the xcex2FGF (basic fibroblast growth factor) of the extracellular matrix, a factor which has been involved in tumoral angiogenesis (Liotta, L. A. et al. (1991) Cell, 327-336. Folkman, J. (1995) New England J. Med., 333: 1757-1763). At the same time, the TGF-xcex21 factor is also mobilized. It shows opposite effects, thus regulating the angiogenesis process (Mignati, P. and Rifkin, D. B., (1993) Physiolog. Rev., 73: 161-195). The process which has been more intensively studied is the role played by the proteases in invasiveness and metastasis. A feature of the invasive processes, either those which take place in the primary tumor or those related to the establishment of metastasis, is the degradation of the extracellular matrix as a result of which the tumoral cells can penetrate the adjacent tissues (the main components of the extracellular matrix are proteins and proteoglycans) (Hynes, R. O. (1994) Bioessays, 16:663-669). In fact, there is a positive relationship between the level of expression of proteases and the aggressivity and malignancy of the tumor due to the greater invasiveness of the tissues of the tumoral cell. These proteases are not specific to the tumoral cells but are also produced by normal cells during the normal recomposition processes of the tissue (wound healing, morphogenesis, etc). The difference is that the tumoral cells links this proteolysis with the motility resulting in an invasion in times and sites which are inappropriate.
Due to the important role which is played by proteases in the tumoral processes and in practically all their stages (concretely, in transformation, invasiveness, adherence and metastasis), various studies have been published in which the possible role of the protease inhibitors as antitumoral agents is analysed (Troll, W. and Kennedy, A. R., editors xe2x80x9cProtease inhibitors as cancer chemopreventive agentsxe2x80x9d, 1993). Protease inhibitors would employ their antitumoral properties at various levels among which we may mention:
1. Blocking the transformation of normal cells in tumoral cells, a process in which proteases seem to be involved (Billings, P. C., et al. (1989) Carcinogenesis, 10: 687-691).
2. Blocking the proteolytic cascade involved in the process of invasiveness and metastasis (Kohn, E. C. and Liotta, L. A. (1995) Cancer Res., 55: 1856-1862).
3. Altering the processing of growth factors required for the development of the tumor.
Among the relevant studies in relation to the present invention are the following: Billings, P. C. et al. (1989) Carcinogenesis, 10: 687-691, where it is described that the addition of PCI in low concentrations (5 xcexcg/mL) inhibits in vitro the tumoral transformation induced by irradiation in embryonic cells of mice. However, up to now, any effect of the PCI in the growth and survival of already transformed cells or in tumoral cells, such as it is described in the present invention, cannot be found in scientific literature. On the other hand, Billings, P. C., et al. (1991) Carcinogenesis, 12: 653-657, explain that the chemotrypsin inhibitor 1 of the potato suppresses the transformation provoked by the irradiation in CH3/10T1/2 cells, in vitro. Billings, P. C. et al. (1991) Eur. J. Cancer, 27: 903-908, describe another inhibitor derived from soya, the so called Bowman-Birk (from now on, referred to as xe2x80x9cBBIxe2x80x9d) which, in mice, reduces cancer of the colon induced by dimethylhydrazine and in (1992) Proc. Natl. Acad. Sci. USA, 889: 3120-3124, characterizes the enzyme target of the BBI as a gelatinase. It has also been described that BBI suppresses carcinogenesis in the intestines (Kennedy, A. R. et al. (1996) Cancer Res., 56: 679-682).
Another relevant aspect for the present invention is the role that growth factors play in the tumoral process. The normal cells are dependent on various growth factors to complete their cellular cycle or to get out of phase G0 (quiescence). In tumoral cells, the system of signal transduction of any of these factors is often found to be altered. For instance, the tumoral cell may present an autocrine loop, or an altered receptor which would be active even in the absence of the growth factor. However, tumoral cells always need some growth factor (IGF-I type) for their proliferation and for the development of the tumor so their absence may provoke apoptosis (Aaronson, S. A. (1991) Science, 254: 1146-1153; Thompson, C. B. (1995) Science, 267, 1456-1462). The migration of the tumoral cell may be regulated by autocrine factors (produced and/or secreted by the same cell), paracrine (growth factors such as those of the PDGF, FGF, EGF and IGF families, whose secretion is stimulated by the stroma), normal cells of the invaded tissue and components of the extracellular matrix. Meantime, the latter also acts as a reservoir for some of these factors.
As regards the present invention, the relationship existing between proteases and the mechanism of cellular apoptosis is also relevant. There is increasing evidence which indicates that apoptosis involves the activation of a cascade of proteases which is not surprising given the fact that the dismantling of a cell requires proteolytic processes (for recent reviews, see Kumar, S. and Harvey, N. (1995) FEBS Letters, 375: 169-173; Takahashi, A. and Earnshaw, W. (1996) Curr. Opin. Genet. and Develop., 6: 50-55; Patel, T. et al. (1996) FASEB J., 10: 587-597). The most important control point to initiate the apoptosis process is in the protein, transcription factor, p53. This factor controls four important cellular mechanisms: (a) the stopping of the cellular cycle in G1 to allow (b) the repairing of the DNA provided that it is not too severely damaged; if the damage is excessive, it stimulates (c) the apoptosis, as well as it inhibits (d) the angiogenesis (Bates, S. and Vousden, K. (1996) Curr. Opin. Genet. and Develop., 6: 12-19).
Due to the suppressing effect that the IGF-IR (receptor of growth factor 1 of the insulin type, from now on referred to as xe2x80x9cIGF-IRxe2x80x9d) has on cellular apoptosis, it presents a potentially therapeutic effect on cancer. An IGF-IR decrease at cellular level is cause of massive apoptosis of the tumoral cell in vivo (Resnikoff, M. et al. (1995) Cancer Res., 55: 2463-2469 and 3739-3741). What is more, the surviving cells are eliminated by the host, apparently by the immune system, prompting a regression of the tumor (Baserga, R. (1996) TIBTECH, 14: 150-152). In short, the IGF-IR has unique features that make it a suitable candidate target for antitumoral therapy; in other words, a molecule that would interfere with the IGF-IR would made way for: (a) massive apoptosis of tumoral cells, (b) would inhibit tumorigenicity, (c) would provoke an immune response in the host, and (d) would have a limited effect on the normal cells (Baserga, R. (1996) TIBTECH, 14: 1501xe2x88x9d152). In addition, the IGF-IR would be a more general target than other receptors for growth factors (Baserga, R. (1995) Cancer. Res., 55: 249-252).
The IGF-IR, together with other growth factors, would play a key role in the mechanism of signal transduction. Apparently, many tumoral cells lose the redundancy of signal transduction pathway present in normal cells. This would be the most probable cause of the vulnerability of tumoral cells in front of signal transduction intercepting molecules (Levitzki, A. (1996) Curr. Opin. Cell Biol., 8: 239-244). For this reason, it is very interesting to find molecules that may intercept signal transduction, to be used on their own or in combination with others as antitumoral agents. Some or all of the receptors of growth factors would be a suitable target for intercepting signal transduction pathways.
Finally, another relevant point to take into consideration for the present invention is the structural analogy, topological, previously described between the PCI and the different cellular growth factors of the group of the knotins (Isaacs, N. (1995) Curr. Opin. Struct. Biol., 5: 391-395; Lin, S. et al. (1995) Nature Struct. Biol., 2: 835-837; Sun, P. and Davies, D. (1995) Ann. Rev. Biophys. Biomol. Struct., 24: 269-291). This analogy has made it possible to establish the hypothesis that the antitumoral activity of the PCI may be also totally or partially related to this knotin topology and not only to the protease inhibiting capacity. Thus, the PCI could act as an antagonist of growth factors and would block tumoral growth. As regards this aspect, it deserves to be mentioned that it has been reported that monoclonal antibodies against EGF interacting with heparin block the growth in vitro of some tumors and, therefore, could be used as therapeutic agents (Modjtahedi, H. and Dean, C. (1995) Biochim. Biophys. Res. Commun., 207: 389-397). In the same way, monoclonal antibodies or antisense RNA or genetic ablation or creation of dominant-negative mutants of the IGF-IR have been found to inhibit various types of tumors (for a review, see Baserga, R. (1996) TIBTECH, 14: 150-152).
Patents Relevant to the Present Invention
An exhaustive examination of the data bases on patents has not uncovered anything based on the carboxypeptidase inhibitor (PCI) as an antitumoral medicament, at least in the form and through the mechanisms established in the present invention. There is only a mention of the use of various commercial inhibitors amongst which is the carboxypeptidase inhibitor, found in creams for protection against solar radiation (U.S. Pat. No. 4,906,457). In addition, research carried out on patents in which other protease inhibitors are used has not, in any way, discorvered any other effect other than that of inhibiting proteases. Effects based on the topology of the molecule (knotin) and/or its interaction with various receptors of growth cellular factors have not been identified.
One of the main aims of the present invention is the use of the carboxypeptidase inhibitor (PCI) from potato, whose amino acid sequence of the most studied isoform, the IIa, is:
SEQ ID NO.2
The invention can be extended to other natural isoforms of the PCI from other solanaceous plants as antitumoral agents.
One of the aims of the present invention is to obtain the isoform II1 of the inhibitor of metalocarboxypeptidases (this form is going to be referred to from now on as xe2x80x9cPCIxe2x80x9d, xe2x80x9crPCIxe2x80x9d or xe2x80x9cwtPCIxe2x80x9d, indifferently by means of recombinant DNA techniques and heterologous expression. Other natural variants or variants redesigned by protein engineering or chemically synthesized are also the object of the present invention.
The use of previous natural or artificial variants of the PCI as antitumoral agents is also object of the present invention.
A further object of the present invention is the determination, at cellular and molecular level, of the way in which the inhibitor of metalocarboxypeptidases carries out antitumoral activity.
Finally, it is object of the present invention to establish the structural basis responsible for the antitumoral activity of the PCI which would allow for the latter to be redesigned, as well as the design of other peptidomimetic or organomimetic molecules with better characteristics of formulation, pharmacokinetics and pharmacodynamics for their therapeutic use as antitumoral agents.